PROJECT SUMMARY We will use two human-adapted microbes that cause tremendous morbidity and mortality worldwide to explore the potential of organoid technology to study human-specific gastrointestinal bacterial pathogens. Helicobacter pylori (Hp) chronically infects the stomachs of over 50% of the world population and is the most important risk factor for the development of gastric cancer. Over 700,000 people die annually from gastric cancer. Salmonella enterica serovar Typhi (S. Typhi) infects more than 20 million people yearly and causes 220,000 deaths annually. Both pathogens can persist over long periods of time in the human host, neither have an animal reservoir, and both manipulate the epithelium and the immune system in sophisticated ways. Each is adapted to a different region of the gastrointestinal tract, and thus will serve as important test cases for infection of different types of human organoids (gastric, small intestine, and colon). There are a number of unanswered questions that cannot be properly addressed in animal models. For example, it is unknown whether the injection of the Hp virulence factor CagA into gastric cells promotes gastric cancer. In vitro, Hp and CagA accelerate epithelial cell proliferation, disrupt the intercellular junctions, alter cell polarity, prevent apoptosis, and induce inflammatory responses. We will use human gastric organoids to determine whether Hp injects CagA in the human gastric glands and how it affects proliferating precursor cells in human epithelium. Another unanswered question in the field is how does Salmonella Typhi invade the intestine without causing acute gastroenteritis? To begin addressing these questions we propose to 1) use gastric organoids to model the interaction between Hp and the human gastric epithelium and focus on its effects on precursor/stem cells and 2) ask how does S.Typhi control immune responses in the gut to manipulate inflammatory signaling and avoid causing acute gastroenteritis.